Interstitial PO(2) determination by phosphorescence quenching microscopy.

Area of Science:

• Physiology
• Biomedical Engineering
• Microscopy

Background:

• Accurate measurement of interstitial oxygen tension (PO(2)) is crucial for understanding skeletal muscle physiology and pathophysiology.
• Existing methods for PO(2) measurement can have limitations in spatial resolution and invasiveness.

Purpose of the Study:

• To introduce and validate a novel technique for determining interstitial PO(2) in skeletal muscle using microinjection of a phosphor probe.
• To assess the spatial distribution and temporal dynamics of the phosphor probe within muscle tissue.
• To evaluate the technique's ability to measure PO(2) gradients around microvessels and during muscle contraction.

Main Methods:

• Surgical isolation of the spinotrapezius muscle in Wistar-Kyoto rats.
• Microinjection of a phosphor probe into the skeletal muscle interstitium.
• Phosphorescence quenching microscopy to measure tissue PO(2) at various time points and distances from microvessels.
• Measurement of PO(2) decline during electrically induced muscle contraction (8-Hz).

Main Results:

• The phosphor probe signal became detectable within a 2.5-mm radius 30 minutes post-injection, with signal intensity increasing over time.
• Nonlinear gradients of PO(2) were observed around arterioles and venules, with significant differences noted at distances >16 microm from microvessel centers.
• During 8-Hz contraction, tissue PO(2) dropped significantly from a baseline of 28.3 mmHg to 8.2 mmHg.

Conclusions:

• The microinjection of phosphor probes combined with phosphorescence quenching microscopy is a viable technique for measuring interstitial PO(2) in skeletal muscle.
• This method provides reliable PO(2) values that align with established oxygen microelectrode techniques.
• The technique is capable of resolving spatial PO(2) heterogeneities and dynamic changes during physiological challenges like muscle contraction.